The objective of this research is to undertake a detailed analysis of an under-investigated class of proteins: the mammalian phosphatidylinositol transfer proteins (PITPs). These proteins catalyze the transport of either phosphatidylinositol or phosphatidylcholine, as monomers, between membrane bilayers in vitro. The precise functions of PITPs in mammalian cells, and the mechanisms by which PITPs execute such functions, remain to be elucidated, however. Present evidence suggests that PITPs play central roles in regulating phospholipid-mediated signal transduction pathways that interface. Present evidence suggests that PITPs play central roles in regulating phospholipid-mediated signal transduction pathways that interface with such diverse cellular processes as protein section, photo-transduction, and receptor-mediated signaling. These functional interfaces are of direct relevance to human disease as to two known cases of inherited PITP insufficiency in higher eukaryotes result in dramatic neurodegenerative diseases that involve progressive loss of specific neuronal populations. The available data identify fundamental roles for PITPs in stimulating specific pathways for neuronal survival and this function is broadly consistent with the enrichment of PITPs in neuronal tissues. The research plan represents a comprehensive and multi-disciplinary effort designed to identify the function(s) executed by a specific mammalian PITP isoform (PITPbeta). PITPbeta physically associates with the Golgi complex and is conserved throughout by the metazoans. We will undertake three lines of investigation to discern the function of this protein in mammals and, in particular, the mammalian nervous system. First, we employ sophisticated photobleaching and quantitative imaging techniques to compare the dynamics of PITPbeta in living cells with those of the related PITPalpha isoform, and to identify the determinants that compare the dynamics of PITPbeta in living cells with those of the related PITPalpha isoform, and to identify the determinants that specify the differential localization of these two PITPs. Second, in more directed approaches for defining PITPbeta function, we shall characterize PITPbeta-deficient mice generated by standard homologous gene targeting methods, and will generate fully developed mice induced for central nervous system-specific PITPbeta deficiencies. Finally, we will develop PITPbeta-deficient cell models so that mechanisms of PITPbeta function at the cellular level can be analyzed. All of these studies will be coupled to the use of novel mutant PITPbeta's in complementation experiments designed to address which phospholipid binding/transfer property is relevant to any particular physiological function of PITPbeta. The proposed studies will provide new and fundamental information that will bear directly on the functions of specific PITP isoforms in mammals, and the molecular mechanisms by which PITPs protect the mammalian nervous system from neurodegenerative disease. The Bankaitis laboratory is in a unique position tp undertake the mammalian nervous system from neurodegenerative disease. The Bankaitis laboratory is in a unique position to undertake this line of research as it has developed the requisite genetic and biochemical systems for such comprehensive analyses.